Anti-angptl 3/8 complex antibodies and methods of using the same

ABSTRACT

Angiopoietin-like protein (ANGPTL)3/8 complexes and antibodies are disclosed, where the antibodes bind to and thereby neutralize ANGPTL3/8 complexes. Pharmaceutical compositions also are disclosed that include one or more anti-ANGPTL3/8 complex antibodies herein in a pharmaceutically acceptable carrier. Methods of making and using the same also are disclosed, especially for increase lipoprotein lipase activity and lowering triglycerides. In this manner, the compounds and compositions may be used in treating lipid metabolism-related and glucose metabolism-related diseases and disorders.

The disclosure relates generally to biology and medicine, and moreparticularly it relates to antibodies (Abs) that bind to and therebyneutralize human angiopoietin-like protein (ANGPTL) 3/8 complexes. SuchAbs can increase lipoprotein lipase (LPL) activity and thereby lowerserum triglycerides (TGs) such that they may be used in treating lipidmetabolism-related and glucose metabolism-related diseases anddisorders.

ANGPTLs are a family of proteins that regulate a number of physiologicaland pathophysiological processes. Of particular interest herein, is therole of ANGPTL3 and ANGPTL8 in lipid and glucose metabolism.

Evidence supports the role of ANGPTL3 as a main regulator of lipoproteinmetabolism, and it may regulate TG clearance by inhibiting LPL andinhibiting endothelial lipase (EL). See, Chi et al. (2017) Mol. Metab.6:1137-1149. ANGPTL3 deficiency, inactivation, or loss can result in lowlevels of low-density lipoprotein-cholesterol (LDL-C), high-densitylipoprotein-cholesterol (HDL-C) and TGs. ANGPTL3 also may affect insulinsensitivity, thereby playing a role in modulating not only lipidmetabolism but also glucose metabolism. See, Robciuc et al. (2013)Arterioscler. Thromb. Vasc. Biol. 33:1706-1713. Nucleic acid and aminoacid sequences for human ANGPTL3 are known. For example, one nucleicacid sequence can be found in NCBI Reference Sequence No. NM_014495 (SEQID NO:1), and one amino acid sequence can be found in NCBI ReferenceSequence No. NP_055310 (SEQ ID NO:2).

ANGPTL8 is highly expressed in the liver and adipose tissue and has beenreported to inhibit LPL by complexing with and thereby activatingANGPTL3. See, Chi, supra. Human ANGPTL8 appears to be induced byfeeding. Nucleic acid and amino acid sequences for human ANGPTL8 areknown. For example, one nucleic acid sequence can be found in NCBIReference Sequence No. NM_018687 (SEQ ID NO:3), and one amino acidsequence can be found in NCBI Reference Sequence No. NP_061157 (SEQ IDNO:4).

ANGPTL3/8 complexes exist, which have one or more ANGPTL3s that arebound to one or more ANGPTL8s. Evidence suggests these complexes moreeffectively mediate inhibition of LPL when compared to ANGPTL3 orANGPTL8 alone. Moreover, ANGPTL3/8 complexes may be made in vitro byco-expressing ANGPTL8 and ANGPTL3 in a mammalian expression system. See,Chi, supra.

Abs are known that bind to either ANGPTL3 or ANGPTL8 and that can beused alone or in combination with each other to treat lipidmetabolism-related and glucose metabolism-related diseases anddisorders. For example, Intl. Patent Application Publication No. WO2012/174178 discloses a fully human monoclonal Ab and antigen-bindingfragments thereof that bind to ANGPTL3 and interfere with its activity.Other therapeutic anti-ANGPTL3 Abs also are known. See, e.g., Intl.Patent Application Publication No. WO 2008/073300 and U.S. Pat. No.7,935,796. Likewise, Intl. Patent Application Publication No. WO2017/027316 discloses a fully human monoclonal Ab or antigen-bindingfragments thereof that bind to ANGPTL8 and interfere with its activity.Moreover, Intl. Patent Application Publication No. WO 2017/177181discloses a combined anti-ANGPTL3 Ab and anti-ANGPTL8 Ab therapy.

Unfortunately, existing Abs that bind only to either ANGPTL3 or ANGPTL8do not fully abrogate the effect of these ANGPTLs and/or ANGPTL3/8complexes on lipid and/or glucose metabolism. See, e.g., Dewey et al.(2017) N. Engl. J. Med. 377:211-221; and Gusarova et al. (2017)Endocrinology 158:1252-1259. In view thereof, there is a need foradditional Abs, especially anti-ANGPTL3/8 complex Abs, for treatinglipid metabolism-related and glucose metabolism-related diseases anddisorders, where such Abs have improved pharmacological inhibitoryand/or regulatory properties to modulate lipid and/or glucosemetabolism.

To address this need, nucleic and amino acid sequences are provided fora modified ANGTPL3/8 complex. Accordingly, nucleic acid sequencesencoding one or more of a modified ANGPTL3 and a modified ANGPTL8 (i.e.,fusion proteins) are described herein. In some instances, the nucleicacid sequences include a polynucleotide sequence encoding an ANGPTL3fusion protein having an amino acid sequence of SEQ ID NO:17. In otherinstances, the nucleic acid sequences include a polynucleotide sequenceencoding an ANGPTL8 fusion protein having an amino acid sequence of SEQID NO:18. In still other instances, the nucleic acid sequences include apolynucleotide sequence encoding SEQ ID NO:17 and 18.

Additionally, nucleic acid constructs are provided that include apolynucleotide sequence encoding an ANGPTL3 fusion protein as describedherein, an ANGPTL8 fusion protein as described herein, or both, wheresuch constructs can be an expression cassette or a vector.

In view of the above, host cells are provided that include therein oneor more expression cassettes or vectors as described herein. In someinstances, the host cells are eukaryotic cells. In some instances, thepolynucleotide sequences for the ANGPTL3 and ANGTPL8 fusion proteins areon separate expression cassettes or vectors, while in other instancesthey can be on the same expression cassette or vector.

Also, ANGPTL3 fusion proteins are provided that include an amino acidsequence of SEQ ID NO:17 or 19, as well as active variants or fragmentsthereof. Likewise, ANGPTL8 fusion proteins are provided that include anamino acid sequence of SEQ ID NO:18 or 20, as well as active variants orfragments thereof.

Moreover, functional ANGPTL3/8 complexes are provided, especially humanANGPTL3/8 complexes, where an ANGPTL3 moiety of the complex is a native(full-length or truncated) ANGPTL3 or an ANGPTL3 fusion protein asdescribed herein and where an ANGPTL8 moiety of the complex is anANGPTL8 fusion protein as described herein. In some instances, theANGPTL3 fusion protein includes an amino acid sequence of SEQ ID NO:19.Likewise, and in some instances, the ANGPTL8 fusion protein includes anamino acid sequence of SEQ ID NO:20. Moreover, and in some instances,the complexes can have a 1:1 ratio of the ANGPTL3 moiety to the ANGPTL8moiety. In other instances, the complexes can have ratios other than1:1, such as 1:2, 1:3, 2:1 or 3:1 ratio of ANGPTL3 moiety to ANGPTL8moiety, respectively.

Methods also are provided for making recombinant ANGTPL3/8 complexes.The methods can include at least a step of expressing one or morepolynucleotide sequences for an ANGPTL3 moiety and an ANGPTL8 moiety asdescribed herein in a host cell such as in a mammalian expression systemto obtain ANGPTL3/8 complexes therefrom. In some instances, the ANGPTL3and ANGPTL8 moieties are provided on separate expression constructs orvectors. In other instances, ANGPTL3 and ANGPTL8 are provided on oneexpression construct or vector. The methods also can include a step ofpurifying the resulting ANGPTL3/8 complexes, which may include not onlyconcentrating the ANGPTL3/8 complexes but also removing one or more ofthe tags, linkers and serum albumin from the ANGPTL3 moiety and/or theANGPTL8 moiety. The methods also can include a step of concentrating theANGPTL3/8 complexes before and/or after purifying step.

Second, an Ab to a ANGPTL3/8 complex is provided as well as usesthereof, which includes treating lipid metabolism-related and glucosemetabolism-related diseases and disorders by binding to and therebyinhibiting ANGPTL3/8 complex activity.

An effective amount of the anti-ANGPTL3/8 complex Ab described herein,or a pharmaceutically acceptable salt thereof, may be used forincreasing LPL activity, lowering TGs, and treating lipid metabolism-and/or glucose metabolism-related diseases or disorders in an individualin need thereof.

The anti-ANGPTL3/8 complex Ab described herein binds soluble ANGPTL3/8complex, thereby increasing LPL activity and decreasing serum TG levels.Individuals with lower TG levels are at lower risk for cardiovasculardisease. Advantageously, the anti-ANGPTL3/8 complex Ab described hereinbinds only to the ANGPTL3/8 complex and not to ANGPTL3 alone or toANGPTL8 alone at relevant concentrations. It is believed that theanti-ANGPTL3/8 complex Ab increases catabolism of TG-rich lipoproteins(TRLs), which reduces TGs and/or non-HDL-C, thereby improvingdyslipidemia risk factors for atherosclerotic cardiovascular disease(ASCVD) not addressed by current therapies. Moreover, and because theanti-ANGPTL3/8 complex Ab described herein does not bind ANGPTL3 orANGPTL8 alone, other actions of these ANGPTLs are not inhibited, whichcan lead to fewer untoward in vivo effects, such as reducedde-repression of EL.

In particular, the anti-ANGPTL3/8 complex Ab is a human anti-ANGPTL3/8complex Ab. In some instances, the anti-ANGPTL3/8 complex Ab canabrogate, block, inhibit, interfere, neutralize or reduce in vivoactivity of the ANGPTL3/8 complex, especially its LPL inhibitoryactivity. In some instances, the anti-ANGPTL3/8 complex Ab can befull-length or can be only an antigen-binding fragment (e.g., a Fab,F(ab′)2 or scFv fragment). Desirable properties of an anti-ANGPTL3/8complex Ab include TG lowering at low doses of the Ab, that is durablefor at least 21 days.

In some instances, the anti-ANGPTL3/8 complex Ab binds human ANGPTL3/8complex and includes light chain determining regions LCDR1, LCDR2 andLCDR3 and heavy chain determining regions HCDR1, HCDR2 and HCDR3, whereLCDR1 has the amino acid sequence RSSQSLLDSDDGNTYLD (SEQ ID NO:11),LCDR2 has the amino acid sequence YMLSYRAS (SEQ ID NO:12) and LCDR3 hasthe amino acid sequence MQRIEFPLT (SEQ ID NO:13), and where HCDR1 hasthe amino acid sequence TFSGFSLSISGVGVG (SEQ ID NO:14), HCDR2 has theamino acid sequence LIYRNDDKRYSPSLKS (SEQ ID NO:15) and HCDR3 has theamino acid sequence ARTYSSGWYGNWFDP (SEQ ID NO:16).

Further provided is an Ab including a light chain variable region(LCVR), where the LCVR has the amino acid sequence of SEQ ID NO:9; or anAb including a heavy chain variable region (HCVR), where the HCVR hasthe amino acid sequence of SEQ ID NO:10. In some instances, the Abincludes an LCVR with the amino acid sequence of SEQ ID NO:9 and a HCVRwith the amino acid sequence of SEQ ID NO:10. In some instances, the Abincludes a light chain (LC) and a heavy chain (HC), where the LC has theamino acid sequence of SEQ ID NO:5 or the HC has the amino acid sequenceof SEQ ID NO:6. Alternatively, the Ab includes a light chain (LC) and aheavy chain (HC), where the LC has the amino acid sequence of SEQ IDNO:5 and the HC has the amino acid sequence of SEQ ID NO:6. In certaininstances, the Ab is an IgG4 isotype.

In some instances, the anti-ANGPTL3/8 complex Ab can be a variant of theAb described above, especially a LC variant having a D31S mutation (SEQID NO:21), a D33A mutation (SEQ ID NO:22), a D33T mutation (SEQ IDNO:23); a M56T mutation (SEQ ID NO:24), a E99Q mutation (SEQ ID NO:25)or a combination thereof (e.g., D33T and M56T mutations or D33A and M56Tmutations), with respect to a LC having an amino acid sequence of SEQ IDNO:5.

Furthermore, an Ab is provided that is produced by cultivating amammalian cell including a cDNA molecule, where the cDNA moleculeencodes polypeptides having the amino acid sequences of SEQ ID NO:5 and6, under such conditions that the polypeptides are expressed, andrecovering the Ab. Alternatively, an Ab is provided that is produced bycultivating a mammalian cell including two cDNA molecules, where a firstcDNA molecule encodes a polypeptide having the amino acid sequence ofSEQ ID NO:5, and a second cDNA molecule encodes a polypeptide having theamino acid sequence of SEQ ID NO:6, under such conditions that thepolypeptides are expressed, and recovering the Ab. In some instances,the anti-ANGPTL3/8 complex Ab can be a variant of the Ab describedabove, especially a LC variant having a D31S mutation (SEQ ID NO:21), aD33A mutation (SEQ ID NO:22), a D33T mutation (SEQ ID NO:23); a M56Tmutation (SEQ ID NO:24), a E99Q mutation (SEQ ID NO:25) or a combinationthereof, with respect to a LC having an amino acid sequence of SEQ IDNO:5.

Moreover, an Ab is provided that binds to and neutralizes humanANGPTL3/8 complex in a standard LPL activity assay with an EC₅₀ of 0.5nM or less. Also, an Ab is provided that binds to human ANGPTL3/8complex with a dissociation constant of less than or equal to 1×10⁻¹ M.Moreover, an Ab is provided that binds to human ANGPTL3 and humanANGPTL8 with a dissociation constant of greater than 1×10⁻¹ M.Furthermore, an Ab is provided that binds to human ANGPTL3/8 complexwith a signal greater than 3 fold over the non-binding backgroundsignal, as measured by single point ELISA assay, but does not bind tohuman ANGPTL3 alone or human ANGPTL8 alone with a signal greater than 3fold over the non-binding background signal, as measured by single pointELISA assay. Likewise, an Ab is provided that lowers TGs in vivo by atleast 50% when compared to IgG control at a dose of 10 mg/kg at a timepoint 14 days after dosing.

Third, a pharmaceutical composition is provided that includes the Abherein or a population of Abs herein and an acceptable carrier, diluentor excipient. Also provided is a mammalian cell including a DNA moleculeincluding a polynucleotide sequence encoding polypeptides having aminoacid sequences of SEQ ID NO:5 and SEQ ID NO:6, where the cell is capableof expressing an Ab herein. Further provided is a mammalian cellincluding a first DNA molecule and a second DNA molecule, where thefirst DNA molecule includes a polynucleotide sequence encoding apolypeptide having the amino acid sequence of SEQ ID NO:5, and where thesecond DNA molecule includes a polynucleotide sequence encoding apolypeptide having the amino acid sequence of SEQ ID NO:6, and where thecell is capable of expressing an Ab herein. In some instances, theanti-ANGPTL3/8 complex Ab can be a variant of the Ab described above,especially a LC variant having a D31S mutation (SEQ ID NO:21), a D33Amutation (SEQ ID NO:22), a D33T mutation (SEQ ID NO:23); a M56T mutation(SEQ ID NO:24), a E99Q mutation (SEQ ID NO:25) or a combination thereof,with respect to a LC having an amino acid sequence of SEQ ID NO:5.

Fourth, a process is provided for producing an Ab, where the processincludes cultivating a mammalian cell with a DNA molecule having apolynucleotide sequence encoding polypeptides having the amino acidsequences of SEQ ID NO:5 and SEQ ID NO:6, where the cell is capable ofexpressing the Ab herein under conditions such that the Ab is expressed,and recovering the expressed Ab. In some instances, the polynucleotidesequence encoding the polypeptide having the amino acid sequence of SEQID NO:5 can encode a D31S mutation, a D33A mutation, a D33T mutation, aM56T mutation, a E99Q mutation or a combination thereof. Also providedherein is a method of treating ASCVD, chronic kidney disease (CK_(D)),diabetes, hypertriglyceridemia, nonalcoholic steatohepatitis (NASH),obesity, or a combination thereof, where the method includesadministering to an individual in need thereof, an effective amount ofan Ab herein. Further provided is a method of lowering TGs that includesadministering to an individual in need thereof, an effective amount ofan Ab herein.

Fifth, an Ab is provided for use in therapy. In particular, the Ab isfor use in the treatment of ASCVD, CKD, diabetes, hypertriglyceridemia,NASH, obsesity, or a combination thereof. Also provided is apharmaceutical composition for use in treating ASCVD, CKD, diabetes,hypertriglyceridemia, NASH, obsesity, or a combination thereof, thatincludes an effective amount of the an the Ab herein.

The advantages, effects, features and objects other than those set forthabove will become more readily apparent when consideration is given tothe detailed description below. Such detailed description makesreference to the following drawing(s), where:

FIG. 1 shows an image of a SDS-page gel showing ANGPTL3/8 complex rununder reduced and non-reduced conditions.

Reference to an element by the indefinite article “a” or “an” does notexclude the possibility that more than one element is present, unlessthe context clearly requires that there be one and only one element. Theindefinite article “a” or “an” thus usually means “at least one.”

Definitions

As used herein, “about” means within a statistically meaningful range ofa value or values such as, for example, a stated concentration, length,molecular weight, pH, sequence similarity, time frame, temperature,volume, etc. Such a value or range can be within an order of magnitudetypically within 20%, more typically within 10%, and even more typicallywithin 5% of a given value or range. The allowable variation encompassedby “about” will depend upon the particular system under study, and canbe readily appreciated by one of skill in the art.

As used herein, “affinity” means a strength of an Ab's binding to anepitope on an ANGPTL3/8 complex.

As used herein, “angiopoietin-like protein 3” or “ANGPTL3” means aprotein having an amino acid sequence including SEQ ID NO:2.

As used herein, “angiopoietin-like protein 8” or “ANGPTL8” means aprotein having an amino acid sequence including SEQ ID NO:4.

As used herein, “ANGPTL3/8 complex” means a multi-protein complex of oneor more ANGPTL3 compounds that are bound to one or more ANGPTL8compounds.

As used herein, “anti-ANGPTL3/8 complex Ab” or “anti-ANGPTL3/8 complexAb” means an Ab that simultaneously recognizes and binds to an area onboth ANGPTL3 and ANGPTL8, especially when in the form of the ANGPTL3/8complex. Generally, an anti-ANGPTL3/8 complex Ab will usually not bindto other ANGPTL family members (e.g., ANGPTL1, ANGPTL2, ANGPTL4,ANGPTL5, ANGPTL6 or ANGPTL7). Moreover, and as noted elsewhere, ananti-ANGPTL3/8 complex Ab also will not bind to ANGPTL3 or ANGPTL8 aloneat specified concentrations, as described in the single point ELISAassay below.

As used herein, “bind” or “binds” means an ability of a protein to forma type of chemical bond or attractive force with another protein ormolecule as determined by common methods known in the art. Binding canbe characterized by an equilibrium dissociation constant (K_(D)) ofabout 1×10⁻⁶ M or less (i.e., a smaller K_(D) denotes a tighterbinding). Methods of determining whether two molecules bind are wellknown in the art and include, for example, equilibrium dialysis, surfaceplasmon resonance, and the like. Here, an anti-ANGPTL3/8 complex Abbinds only the ANGPTL3/8 complex and does not bind ANGPTL3 alone orANGPTL8 alone. Whether an Ab binds only to the ANGPTL3/8 complex and notto ANGPTL3 alone or ANGPTL8 alone can be determined in standard ELISAassays in a single point format, as described below and binding may becharacterized by Biacore, as described below. While the Abs herein arehuman, they may, however, exhibit cross-reactivity to other ANGPTL3/8complexes from other species, for example, cynomolgus monkey ANGPTL3/8complex, mouse ANGPTL3/8 complex, or rat ANGPTL3/8 complex.

As used herein, “effective amount” means an amount or dose of a compoundor a pharmaceutical composition containing the same, which upon singleor multiple dose administration to an individual, will elicit abiological or medical response of or desired therapeutic effect on atissue, system, animal, mammal or human that is being sought by theresearcher, veterinarian, medical doctor or other clinician. In someinstances, an effective amount of a compound herein or compositionsincluding the same to an individual in need thereof would result inincreasing LPL activity. A dose can include a higher initial loadingdose, followed by a lower dose. A dose can be administered in anytherapeutically effective interval, such as multiple times a day, oncedaily, every other day, three times a week, two times a week, one time aweek, once every two weeks, once a month, once every two months, etc. Adose constituting an effective amount could be between 0.01 mg/kg and100 mg/kg.

As used herein, “equilibrium dissociation constant” or “K_(D)” means aquantitative measurement of Ab affinity to a particular antigeninteraction, such as affinity of an Ab to ANGPTL3/8 complex, especiallya measure of a propensity of an Ab/ANGPTL3/8 complex conjugate toseparate reversibly into its component parts. Likewise, and as usedherein, “equilibrium association constant” or “K_(a)” means an inverseof KID.

As used herein, “functional” means that and ANGPTL3 fusion protein,ANGPTL8 fusion protein or ANGPTL3/8 complex has biological activity akinto that of a native ANGPTL3, a native ANGPTL8 or a native ANGPTL3/8complex including, for example, inhibiting LPL or acting as an antigento which an Ab can be made and directed.

As used herein, “glucose metabolism-related disease or disorder” meansdiabetes and the like.

As used herein, “lipid metabolism-related disease or disorder” means acondition associated with abnormal lipid metabolism such asdyslipidemia, hyperlipidemia and hyperlipoproteinemia, includinghypertriglyceridemia, hypercholesterolemia, chylomicronemia, mixeddyslipidemia (obesity, metabolic syndrome, diabetes, etc.),lipodystrophy and lipoatrophy. The term also encompasses certaincardiovascular diseases such as atherosclerosis and coronary arterydisease, acute pancreatitis, NASH, obesity and the like.

As used herein, “half-maximal effective concentration” or “EC₅₀” means aconcentration of an Ab (typically expressed in molar units (M)) thatinduces a response halfway between a baseline and a maximum after apredetermined period of time. The EC₅₀ described herein is ideally 3.0nM or less.

As used herein, “nucleic acid construct” or “expression cassette” meansa nucleic acid molecule having at least a control sequence operablylinked to a coding sequence. In this manner, a control sequence such asa promoter is in operable interaction with nucleic acid sequencesencoding at least one polypeptide of interest such as the ANGPTL3 fusionproteins described herein and/or the ANGPTL8 fusion proteins describedherein. Such nucleic acid constructs can be in the form of an expressionor transfer cassette. A nucleic acid construct can include anoligonucleotide or polynucleotide composed of deoxyribonucleotides,ribonucleotides or combinations thereof having incorporated therein thenucleotide sequences for one or more polypeptides of interest.

As used herein, “operably linked” means that the elements of a nucleicacid construct are configured so as to perform their usual function.Thus, control sequences (i.e., promoters) operably linked to a codingsequence are capable of effecting expression of the coding sequence. Thecontrol sequences need not be contiguous with the coding sequence, solong as they function to direct the expression thereof (i.e., maintainproper reading frame). Thus, for example, intervening untranslated, yettranscribed sequences, can be present between a promoter and a codingsequence, and the promoter sequence still can be considered “operablylinked” to the coding sequence.

As used herein, “control sequence” or “control sequences” meanspromoters, polyadenylation signals, transcription and translationtermination sequences, upstream regulatory domains, origins ofreplication, internal ribosome entry sites (“IRES”), enhancers, and thelike, which collectively provide for replication, transcription andtranslation of a coding sequence in a recipient host cell. Not all ofthese control sequences need always be present so long as the selectedcoding sequence is capable of being replicated, transcribed andtranslated in an appropriate host cell.

As used herein, “coding sequence” or “coding sequences” means a nucleicacid sequence that encodes for one or more polypeptides of interest, andis a nucleic acid sequence that is transcribed (in the case of DNA) andtranslated (in the case of RNA) into a polypeptide in vitro or in vivowhen placed under the control of appropriate regulatory sequences. Theboundaries of the coding sequence(s) are determined by a start codon ata 5′ (amino) terminus and a translation stop codon at a 3′ (carboxy)terminus. A coding sequence can include, but is not limited to, viralnucleic acid sequences, cDNA from prokaryotic or eukaryotic mRNA,genomic DNA sequences from prokaryotic or eukaryotic DNA, or evensynthetic DNA sequences.

With respect to control and coding sequences, they can benative/analogous to the host cell or each other. Alternatively, thecontrol and coding sequences can be heterologous to host cell or eachother.

As used herein, “promoter” means a nucleotide region composed of anucleic acid regulatory sequence, where the regulatory sequence isderived from a gene or synthetically created and is capable of bindingRNA polymerase and initiating transcription of a downstream(3′-direction) coding sequence. A number of promoters can be used innucleic acid constructs, including a native promoter for one or morepolypeptides of interest. Alternatively, promoters can be selected basedupon a desired outcome. Such promoters can include, but are not limitedto, inducible promoters, repressible promoters and constitutivepromoters.

As used herein, “variant” means a polynucleotide or a polypeptide havingone or more modifications such as an addition, deletion, insertionand/or substitution of one or more specific nucleic acid or amino acidresidues when compared to a reference nucleic acid or amino acidsequence. A variant therefore includes one or more alterations whencompared to the reference nucleic acid or amino acid sequence. Here, theanti-ANGPTL3/8 complex Ab can have a LC or HC variation. In particular,the Ab can be a LC variant having a D31S mutation (SEQ ID NO:21), a D33Amutation (SEQ ID NO:22), a D33T mutation (SEQ ID NO:23); a M56T mutation(SEQ ID NO:24) or a E99Q mutation (SEQ ID NO:25) with respect to a LChaving an amino acid sequence of SEQ ID NO:5. Likewise, the LC variantcan be a combination any two of the above such as, for example, D31S andD33A mutations, D31S and D33T mutations, D31S and M56T mutations, D31Sand E99Q mutations, D33A and M56T mutations, D33A and E99Q mutations,D33T and M56T mutations, D33T and E99Q mutations, and M56T and E99Q,again with respect to a LC having an amino acid sequence of SEQ ID NO:5.Moreover, the LC variant can be a combination of any three of the abovesuch as, for example, D31S, D33A and M56T mutations, D31S, D33A and E99Qmutations, D31S, D33T and M56T mutations, D31S, D33T and E99Q mutaitons,D33A, M56T and E99Q mutations, and D33T, M56T and E99Q mutations, againwith respect to a LC having an amino acid sequence of SEQ ID NO:5.Furthermore, the LC variant can be a combination of any four of theabove such as, for example, D31S, D33A, M56T and E99Q mutations; andD31S, D33T, M56T and E99Q mutations, again with respect to a LC havingan amino acid sequence of SEQ ID NO:5.

As used herein, “vector” means a replicon, such as a plasmid, phage orcosmid, to which another nucleic acid sequence, such as an expressioncassette, may be attached so as to bring about replication of theattached sequence. A vector is capable of transferring nucleic acidmolecules to host cells. Vectors typically include one or a small numberof restriction endonuclease recognition sites where a nucleotidesequence of interest can be inserted in a determinable fashion withoutloss of essential biological function of the vector, as well as aselectable marker that can be used for identifying and selecting hostcells transformed with the vector. A vector therefor can be capable oftransferring nucleic acid sequences to target cells.

As used herein, “treatment” or “treating” means management and care ofan individual having a condition for which anti-ANGPTL3/8 complex Abadministration is indicated for the purpose of combating or alleviatingsymptoms and complications of those conditions. Treating includesadministering a compound or compositions containing a compound herein tosuch an individual to prevent the onset of symptoms or complications,alleviating the symptoms or complications, or eliminating the disease,condition, or disorder. Treating includes administering a compound orcompositions containing a compound herein to an individual in needthereof to result in increasing LPL activity and lowering of TGs. Theindividual to be treated is an animal, espeically a human being.

As used herein, “patient,” “subject” and “individual,” are usedinterchangeably herein, and mean an animal, espeically a human. Incertain instances, the individual is a human and is furthercharacterized with a disease, disorder or condition that would benefitfrom administration of an anti-ANGPTL3/8 complex Ab.

As used herein, “antibody” or “Ab” and the like means a full-length Abincluding two heavy chains and two light chains having inter- andintra-chain disulfide bonds. The amino-terminal portion of each of thefour polypeptide chains includes a variable region primarily responsiblefor antigen recognition. Each HC includes an N-terminal HCVR and an HCconstant region (HCCR). Each light chain includes a light chain (LC)variable region (LCVR) and a LC constant region (LCCR). Here, the Ab isan immunoglobulin G (IgG) type Ab, and the IgG isotype may be furtherdivided into subclasses (e.g., IgG1, IgG2, IgG3 and IgG4). The HCVR andLCVR regions can be further subdivided into regions ofhyper-variability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each HCVR and LCVR includes three CDRs and four FRs,arranged from N-terminus to C-terminus in the following order: FR1,CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the three CDRs of the HC arereferred to as HCDR1, HCDR2 and HCDR3, and the three CDRs of the LC arereferred to as LCDR1, LCDR2 and LCDR3. The CDRs contain most of theresidues that form specific interactions with an antigen, such as anANGPTL3/8 complex. Assigning the residues to the various CDRs may bedone by algorithms such as, for example, Chothia, Kabat or North. TheNorth CDR definition is based on affinity propagation clustering with alarge number of crystal structures (North et al. (2011) J. Mol. Bio.406:228-256). Herein, the CDRs are best defined by the sequences listedin the Sequence Listing, which are based upon a combination of multipledefinitions including North.

An isolated DNA encoding a HCVR region can be converted to a full-lengthheavy chain gene by operably linking the HCVR-encoding DNA to anotherDNA molecule encoding heavy chain constant regions. Sequences of human,as well as other mammalian, heavy chain constant region genes are knownin the art. DNA fragments encompassing these regions can be obtained by,for example, standard PCR amplification.

An isolated DNA encoding a LCVR region may be converted to a full-lengthlight chain gene by operably linking the LCVR-encoding DNA to anotherDNA molecule encoding a light chain constant region. Sequences of human,as well as other mammalian, light chain constant region genes are knownin the art. DNA fragments encompassing these regions can be obtained bystandard PCR amplification. The light chain constant region can be akappa or lambda constant region.

The Abs herein may contain an IgG4-PAA Fc portion. The IgG4-PAA Fcportion has a Ser to Pro mutation at position 231 (S231P), a Phe to Alamutation at position 237 (F237A), and a Leu to Ala mutation at position238 (L238A) as numbered by absolute position in SEQ ID NO:6. The S231Pmutation is a hinge mutation that prevents half-Ab formation (phenomenonof dynamic exchange of half-molecules in IgG4 Abs). The F237A and L238Amutations further reduce effector function of the already low human IgG4isotype. It is contemplated, however, that the Abs herein mayalternative include a different Fc portion.

To reduce the potential induction of an immune response when dosed inhumans, certain amino acids may require back-mutations to match Abgermline sequences.

Pharmaceutical compositions including the compounds herein may beadministered parenterally to individuals in need of such treatment. Suchindividual may have ASCVD or be at high risk for ASCVD. Theseindividuals may have acute coronary syndromes, history of myocardialinfarction (MI), stable or unstable angina, coronary or other arterialrevascularization, stroke, transient ischemic attack (TIA), thoracic orabdominal aortic aneurysm, or peripheral arterial disease presumed to beof atherosclerotic origin. Individuals at high risk for ASCVD mayfurther have type 2 diabetes (T2D), CKD or familial hypercholesterolemia(FH).

Parenteral administration may be performed by subcutaneous,intramuscular or intravenous injection by means of a syringe, optionallya pen-like syringe, or mechanical driven injector. Alternatively,parenteral administration can be performed by means of an infusion pump.In some instances, pharmaceutical compositions suitable foradministration to an individual having a therapeutically effectiveamount of a compound oherein and one or more pharmaceutically acceptableexcipients. Such pharmaceutical compositions may be prepared by any of avariety of techniques using conventional excipients for pharmaceuticalproducts that are well known in the art. See, e.g., Remington, “TheScience and Practice of Pharmacy” (D. B. Troy ed., 21^(st) Ed.,Lippincott, Williams & Wilkins, 2006).

The compounds herein may be used in simultaneous, separate or sequentialcombination with one or more additional therapeutic agents useful formodulating LPL activity, treating lipid metabolism-related diseases ordisorders, or treating glucose metabolism-related diseases or disorders,including any of the disorders listed above. Non-limiting examples ofthe additional therapeutic agents that can be combined with the claimedcompounds include, but are not limited to, anti-diabetic agents such asinsulin or insulin analogs, biguanides, sulfonylureas,thiazolidinediones, dipeptidyl peptidase-4 (“DPP-4”) inhibitors, orsodium-dependent glucose transporter (SGLT2) inhibitors; incretincompounds such as glucagon-like-peptide-1 (GLP-1) or GLP-1 analogs,gastric inhibitory polypeptide (GIP) or GIP analogs, oxyntomodulin (OXM)or OXM analogs; aspirin; antiplatelet agents; H2 receptor blockers;proton pump inhibitors; antihypertensives; lipid modifying therapiessuch as HMG-CoA reductase inhibitors, PCSK9 inhibitors, cholesterolabsorption inhibitors, fibrates, niacin, LXR agonists, RXR agonists, RORagonists, or reverse cholesterol transport modulators; heart failuretherapies such as ACE, angiotensin receptor neprilysin inhibitors, ARB,or B adrenergic antagonists; anti-inflammatory therapies; hypertensiontherapies, atrial fibrillation therapies; neurodegeneration therapies;oncology therapies; therapies for diabetic cardiomyopathy, diabeticretinopathy, diabetic neuropathy, diabetic nephropathy, weightreduction, wound healing; nephropathy therapies; PAD therapies, orcombinations of any of the foregoing agents. The anti-ANGPTL3/8 complexAb and the one or more additional therapeutic agent(s) can beadministered either together through the same delivery route and devicesuch as a single pill, capsule, tablet, or injectable formulation; orseparately administered either at the same time in separate deliverydevices or routes; or administered sequentially.

Preparation and Purification of ANGPTL3/8 Complexes

One aspect of the disclosure is ANGPTL3/8 complexes that can be used tomake an Ab that binds to the complex only and do not bind to ANGPTL3alone or to ANGPTL8 alone. Although anti-ANGPTL3 Abs and anti-ANGPTL8Abs are known, there is a challenge in synthesizing sufficientquantities of functional ANGPTL3/8 complexes, especially human ANGPTL3/8complexes, to make anti-ANGPTL3/8 complex Abs. Additionally oralternatively, such ANGPTL3/8 complexes can be used in assays to assessthe properties of Abs directed to ANGPTL3, ANGPTL8 and/or ANGPTL3/8complexes.

In this manner, the disclosure also describes methods of generatingANGPTL8 by making it as either an N- or a C-terminal serum albumin(e.g., human, mouse or rabbit) fusion protein. Functional ANGPTL3/8complexes then can be made by co-expressing the ANGPTL8 fusion proteinwith native ANGPTL3 or an ANGPTL3 fusion protein in a mammalianexpression system.

As noted above, nucleic and amino acid sequences for native, humanANGPTL3 and native, human ANGPTL8 are known (see, e.g., SEQ ID NOS:1-2and 3-4, respectively). ANGPTL3 or ANGPTL 8 as described herein,however, are modified (i.e., recombinant/synthetic) and therefor differfrom the native sequences by including additional amino acid sequencesto improve generating, secreting and/or complexing ANGTPL3 and ANGTPL8.

For example, ANGPTL3 can be modified to include one or more linkers andtags as are known in the art. Here, human ANGPTL3 (SEQ ID NO:2) ismodified to include a linker and FLAG-tag such that the ANGPTL3 fusionprotein has an amino acid sequence of SEQ ID NO:17. In some instances,the linker can be from about 1 to about 10 amino acids, such as 3 aminoacids, especially 3 Ala residues. The linker and FLAG-tag can be placedat the N- or C-terminus of the ANGPTL3 sequence, especially theC-terminus as in SEQ ID NO:17, where residues 1-460 correspond toANGPTL3, and residues 461-471 correspond to the 3-Ala linker andFLAG-tag.

Likewise, ANGPTL8 can be modified to include one or more linkers andtags in addition to a sequence for serum albumin, especially human serumalbumin. Here, human ANGPTL8 (SEQ ID NO:4) is modified to include alinker, an IgG kappa signal peptide, a polyhistidine (His)-tag, maturehuman serum albumin, a linker and a PreScission® Cleavage Site such thatthe ANGPTL8 fusion protein has an amino acid sequence of SEQ ID NO:18.In some instances, the linker can be from about 1 to about 10 aminoacids, such as a rigid polyproline repeat, especially an Ala-Pro (AP)-10linker. The signal peptide, His-tag, mature HSA, linker and PreScission®Cleavage Site can placed at the N- or C-terminus of the ANGPTL8sequence, especially the N-terminus as in SEQ ID NO:18, where residues1-20 correspond to the IgG kappa signal peptide, residues 21-27correspond to the His-tag, residues 28-612 correspond to HSA, residues613-632 correspond to the AP-10 linker, residues 633-643 correspond tothe PreScission® Cleavage Site, and residues 644-820 correspond toANGPTL8.

Methods of constructing nucleic acid constructs to express the ANGPTL3fusion protein and/or the ANGPTL8 fusion protein as described herein arewell known in the art and can be found in, for example, Balbás &Lorence, “Recombinant Gene Expression: Reviews and Protocols” (2^(nd)Ed. Humana Press 2004); Davis et al., “Basic Methods in MolecularBiology” (Elsevier Press 1986); Sambrook & Russell, “Molecular Cloning:A Laboratory Manual” (3^(rd) Ed. Cold Spring Harbor Laboratory Press2001); Tijssen, “Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes” (Elsevier 1993); and“Current Protocols in Molecular Biology” (Ausubel et al. eds., GreenePublishing and Wiley-Interscience 1995); as well as U.S. Pat. Nos.6,664,387; 7,060,491; 7,345,216 and 7,494,805. Because ANGPTL8 does notcontain any disulfide bonds, mammalian expression systems can be used.Here, HEK293 expression systems or CHO expression systems may be used togenerate the ANGPTL3 and/or ANGPTL8 fusion proteins, especially byco-expression.

In addition to expressing the ANGPTL3 fusion protein and/or the ANGPTL8fusion protein, the methods also can include purifying the resultingfusion proteins based upon the particular tag used for each fusionprotein, which are well known in the art. With regard to the ANGPTL3 andANGTPL8 fusion proteins described herein, purification can result in anumber of truncations after removing the tags such that the ANGPTL3fusion protein has an amino acid sequence of SEQ ID NO:19 (whereresidues 1-444 correspond to ANGPTL3, and residues 445-455 correspond tothe 3-Ala linker and FLAG-tag) and the ANGPTL8 fusion protein has anamino acid sequence of SEQ ID NO:20 (where residues 1-4 correspond tocleavage residues from the PreScission® Cleavage Site, and residues5-182 correspond to a fragment of ANGPTL8), which readily associate withone another to form functional ANGPTL3/8 complexes.

Preparation and Purification of Antibodies

Anti-ANGPTL3/8 complex Abs may be produced in a mammalian cellexpression system using a CHO GSKO cell line. A glutamine synthetase(GS) gene knockout enables tightened selection stringency by eliminatingendogenous GS background activity, which can allow survival of low- ornon-productive cells under selection conditions. Genes coding for the AbHC and LC herein may be sub-cloned into individual GS-containingexpression plasmids for co-transfection or both chains may be sub-clonedinto a single GS-containing expression plasmid. The cDNA sequenceencoding the HC or LC is fused in frame with the coding sequence of asignal peptide, which may be the murine kappa leader sequence, toenhance secretion of the desired product into the cell culture medium.The expression is driven by the viral cytomegalovirus (CMV) promoter.

CHO GSKO cells are stably transfected using electroporation and theappropriate amount of recombinant HC and LC expression plasmids, and thetransfected cells are maintained in suspension culture, at the adequatecell density. Selection of the transfected cells is accomplished bygrowth in glutamine-free, 25 μM methionine sulfoximine (MSX)-containingserum-free medium and incubated at 32-37° C. and 5%-7% CO₂. Abs aresecreted into the media from the CHO cells. The Abs may be purified byProtein A affinity chromatography followed by anion exchange, orhydrophobic interaction chromatography (or other suitable methods), andmay utilize size exclusion chromatography for further purification.

Abs from harvested media are captured onto MabSelect SuRe Protein Aresin (GE Healthcare). The resin is then briefly washed with a runningbuffer, such as a phosphate buffered saline (PBS) pH 7.4 or a runningbuffer containing Tris, to remove non-specifically bound material. Absare then eluted from the resin with a low pH solution, such as 20 mMacetic acid/5 mM citric acid. Fractions containing ANGPTL3/8 Abs arepooled and may be held at a low pH to inactivate potential viruses. ThepH can be increased as needed by adding a base such as 0.1 M Tris pH8.0. ANGPTL3/8 Abs may be further purified by hydrophobic interactionchromatography (HIC) using resins such as Phenyl HP (GE Healthcare).Anti-ANGPTL3/8 Abs can be eluted from the HIC column using a sodiumsulfate gradient in 20 mM Tris, pH 8.0. The anti-ANGPTL3/8 Abs may befurther purified by size exclusion chromatography using a Superdex 200column (GE Healthcare) with isocratic elution in PBS, pH 7.4.

The compounds described herein are prepared in this manner or in asimilar manner that would be readily determined by one of skill in theart.

Mice harbouring human variable light and heavy chain domains areimmunized with the ANGPTL3/8 complex described above, and singleantigen-specific B cells are isolated by FACS using ANGPTL3/8 (positive)and ANGPTL3 (negative) as markers. Heavy chain and light chain variableregion DNA are recovered from single B cells by PCR and cloned into IgGexpression vetors. CHO cell supernatants are tested after transfectionfor binding activity.

EXAMPLES

The following non-limiting examples are offered for purposes ofillustration, not limitation.

Example 1 Making ANGPTL3/8 Complexes

ANGPTL3 and ANGPTL8 Expression: nucleotide sequences encoding an aminoacid sequence for human ANGPTL3, a linker and a FLAG tag (SEQ ID NO:17)are inserted into a mammalian expression vector containing a CMVpromoter. Likewise, nucleotide sequences encoding an amino acid sequencefor human ANGTPL8, mouse IgG kappa signal peptide, HIS-tag, mature humanserum albumin (HSA)-PreScission® cleavage site (SEQ ID NO:18) areinserted into a mammalian expression vector containing a CMV promoter.Protein expression is through transient co-transfection of both theabove-described expression vectors in HEK293F cells cultured inserum-free media. Culture media is harvested 5 days post transfectionand is stored at 4° C. for subsequent protein purification.

Protein Purification: protein purification is conducted at 4° C., where4 L culture media are supplemented with 1 M Tris-HCl (pH 8.0) and 5 MNaCl to a final concentration of 25 mM and 150 mM, respectively. Themedia are then incubated with 150 ml of Ni-NTA resin (Qiagen) overnight.The resin is packed into a column and is washed with Buffer A (50 mMTris-HCl, pH 8.0, 0.3 M NaCl). Protein is eluted with 0-300 mM imidazolegradient in Buffer A. Fractions containing HIS-HSA-ANGPTL8/ANGPTL3-Flagare pooled, concentrated and loaded onto a HiLoad® Superdex® 200 Column(GE Healthcare Biosciences), and eluted with Buffer A. Fractionscontaining HIS-HSA-ANGPTL8/ANGPTL3-Flag again are pooled, concentratedand digested with PreScission® Protease (GE Healthcare Biosciences) toremove HSA from HIS-HSA-ANGPTL8 fusion protein. ThePreScission®-digested protein sample is loaded onto a HiLoad® Superdex®200 Column and is eluted with storage buffer (20 mM HEPES, pH8.0, 150 mMNaCl). Fractions containing ANGPTL3/8 complex are pooled andconcentrated, with protein concentration determined using the Bradfordmethod. ANGTPL3/8 complex is aliquoted and stored at −80° C. untilfurther use.

LPL Activity Assay: An EnzCheck® LPL assay is performed according to themanufacture's instructions (ThermoFisher Scientific). Briefly, ANGPTL3and the ANGPLT3/8 complexes are serially diluted in growth medium andreplaced LPL cell medium for 1 hour incubation. EnzCheck® LipaseSubstrate (ELS) is then added into LPL-expressing cells and is incubatedfor 30 minutes. Fluorescence is measured at 482 nm/515 nm(excitation/emission, respectively). The % inhibition of ANGPTL3 and 3/8on LPL are calculated.

Results:

Table 1 shows the yield of ANGTPL3/8 complex protein at the variousstages of purification/concentration.

TABLE 1 ANGPTL3/8 Complex Protein Yield. Ni—NTA Resin 1^(st) Superdex ®200 2^(nd) Superdex ® 200 Start (L) Yield (mg) Column Yield (mg) ColumnYield (mg) 4 105 47 12

Likewise, FIG. 1 shows a 4-20% TG TGX Coomassie-stained gel of thepurified and concentrated ANGPTL3/8 complex resulting from the columns,which confirms that complexes form/assemble. Following purification andconcentration, ANGTL3 has an amino acid sequence of SEQ ID NO:19 andANGPTL8 has an amino acid sequence of SEQ ID NO:20.

In the LPL assay, the EC₅₀ of ANGPTL3 is 21.5 nM while that of the EC₅₀of ANGTPL3/8 complex is 0.54 nM, which confirms that the complexes arefunctional.

Example 2 Assays

Single Point ELISA (SPE) Assay: Ab binding selectivity to either theANGPTL3/8 complex, free ANGPTL3 or free ANGPTL8 is initially verifiedusing standard ELISA assays in a single point format. Briefly, assayplates are coated with an anti-human Fc Ab at a concentration of 2 μg/mland subsequently blocked with casein. IgG secreted into supernatantsafter expression in CHO cells are then captured to the assay plate.Biotinylated antigen is added at a concentration of 25 nM to allow forAb/antigen binding. Ab/antigen complexes are detected after addingalkaline phosphatase-conjugated neutravidin and alkaline phosphatasesubstrate, and subsequent measurement of the optical density at 560 nm.Positive binding is determined by a signal greater than 3 fold over thenon-binding background signal.

ELISA Assay: Ab binding selectivity to either the ANGPTL3/8 complex,free ANGPTL3 or free ANGPTL8 is verified using standard ELISA assays.Briefly, assay plates are coated with an anti-human Fc Ab at aconcentration of 2 μg/ml and subsequently blocked with casein. Ab fromCHO supernatants after expression in CHO cells is then captured to theassay plate and results is an Ab concentration of 2 μg/ml. Biotinylatedantigen (ANGPTL3, ANGPTL8 or ANGPTL3/8 complex) is added at varyingconcentrations by serial dilution to allow for Ab/antigen binding.Ab/antigen complexes are detected after the addition of alkalinephosphatase-conjugated neutravidin, and alkaline phosphatase substrate,and subsequent measurement of the optical density at 560 nm.

TABLE 2 Ab Binding Selectivity to ANGPTL3/8 Complex, ANGPTL8 and ANGPTL3in a SPE Assay. ANGPTL3/8 ANGPTL8 ANGPTL3 Complex Binding BindingBinding Ab 2.302 (positive) 0.087 (negative) 0.074 (negative) D31S 0.9280.072 0.058 D33A 0.898 0.118 0.076 D33T 0.661 0.070 0.060 M56T 0.7750.085 0.060 E99Q 1.538 0.100 0.083 Average 0.09 0.07 0.08 NonbindingBackground Signal 3 × Background 0.27 0.21 0.24 Values are opticaldensity (OD) at 560 nm, and the non-binding background signal across allplates tested on average is reflected in the “Background signal” row.This negative control shows the background signal in the absence of Ab.

In the SPE assay, the Ab having a LC of SEQ ID NO:5 and a HC of SEQ IDNO:6 shows positive binding to the human ANGPTL3/8 complex and negativebinding to human ANGPTL8 and human ANGPTL3. Abs with LC variants havinga D31S mutation (SEQ ID NO:21), a D33A mutation (SEQ ID NO:22), a D33Tmutation (SEQ ID NO:24), a M56T mutation (SEQ ID NO:24) or a E99Qmutation (SEQ ID NO:25) and a HC of SEQ ID NO:6 likewise show binding tothe human ANGPTL3/8 complex and negative binding to human ANGPTL8 andhuman ANGPTL.

TABLE 3 ELISA Assay of Ab Binding to Various Concentrations of ANGPTL3,ANGPTL8 and ANGPTL3/8 Complex. Antigen hANGPTL8 hANGPTL3 hANGPTL3/8complex conc. [nM] Ab Control Ab Control Ab Control 100 0.072 0.0760.081 0.091 2.705 0.063 33.3 0.050 0.060 0.052 0.063 2.411 0.056 11.10.045 0.051 0.046 0.052 1.708 0.047 3.70 0.051 0.048 0.046 0.052 1.0420.046 1.23 0.043 0.048 0.043 0.052 0.510 0.046 0.412 0.046 0.050 0.0430.067 0.231 0.046 0.137 0.047 0.053 0.048 0.055 0.110 0.044 0.046 0.0510.054 0.060 0.069 0.078 0.053 Control is buffer and no Ab.

The Ab having a LC of SEQ ID NO:5 and a HC of SEQ ID NO:6 shows positivebinding to the human ANGPTL3/8 complex in a concentration-dependentmanner and no detectable binding to human ANGPTL8 and human ANGPTL3 upto an antigen concentration of 100 nM in this assay (Table 3).

In addition to the above anti-ANGPTL3/8 complex Ab having a LC of SEQ IDNO:5 and a HC of SEQ ID NO:6, Abs with LC variants having a D31Smutation (SEQ ID NO:21), a D33A mutation (SEQ ID NO:22) or a E99Qmutation (SEQ ID NO:25) and a HC of SEQ ID NO:6 are assayed and likewiseshow positive binding to the human ANGPTL3/8 complex in aconcentration-dependent manner and no detectable binding to humanANGPTL8 and human ANGPTL3 up to an antigen concentration of 100 nM inthis assay (Tables 4-6).

TABLE 4 ELISA Assay of D31S Variant Ab Binding to Various Concentrationsof ANGPTL3, ANGPTL8 and ANGPTL3/8 Complex. Antigen hANGPTL8 hANGPTL3hANGPTL3/8 complex conc. [nM] Ab Control Ab Control Ab Control 100 0.0980.076 0.084 0.081 2.471 0.092 33.3 0.063 0.065 0.069 0.064 2.226 0.06811.1 0.062 0.059 0.062 0.062 1.735 0.053 3.70 0.056 0.055 0.060 0.0571.134 0.049 1.23 0.058 0.049 0.059 0.058 0.575 0.048 0.412 0.062 0.0490.058 0.057 0.234 0.047 0.137 0.080 0.077 0.057 0.076 0.137 0.068 0.0460.096 0.085 0.060 0.064 0.149 0.075

TABLE 5 ELISA Assay of D33A Variant Ab Binding to Various Concentrationsof ANGPTL3, ANGPTL8 and ANGPTL3/8 Complex. Antigen hANGPTL8 hANGPTL3hANGPTL3/8 complex conc. [nM] Ab Control Ab Control Ab Control 100 0.0780.076 0.082 0.081 2.388 0.092 33.3 0.049 0.065 0.067 0.064 2.179 0.06811.1 0.048 0.059 0.060 0.062 1.635 0.053 3.70 0.048 0.055 0.056 0.0571.040 0.049 1.23 0.049 0.049 0.057 0.058 0.491 0.048 0.412 0.047 0.0490.056 0.057 0.196 0.047 0.137 0.053 0.077 0.062 0.076 0.101 0.068 0.0460.074 0.085 0.062 0.064 0.111 0.075

TABLE 6 ELISA Assay of E99Q Variant Ab Binding to Various Concentrationsof ANGPTL3, ANGPTL8, and ANGPTL3/8 Complex. Antigen hANGPTL8 hANGPTL3hANGPTL3/8 complex conc. [nM] Ab Control Ab Control Ab Control 100 0.0980.076 0.084 0.081 2.471 0.092 33.3 0.063 0.065 0.069 0.064 2.226 0.06811.1 0.062 0.059 0.062 0.062 1.735 0.053 3.70 0.056 0.055 0.060 0.0571.134 0.049 1.23 0.058 0.049 0.059 0.058 0.575 0.048 0.412 0.062 0.0490.058 0.057 0.234 0.047 0.137 0.080 0.077 0.057 0.076 0.137 0.068 0.0460.096 0.085 0.060 0.064 0.149 0.075

Example 3 In Vitro Receptor Affinity

Binding kinetics may be determined using a Biacore® T200 Instrument (GEHealthcare Bio-Sciences Corp.; Piscataway, N.J.). A CM4 sensor chipsurface may be prepared by covalent coupling of Human Fab Binder (GEHealthcare Bio-Sciences Corp.). Kinetic experiments may be carried outat about 25° C. in a running buffer of HBSEP+, 0.01% BSA at pH 7.4. Absmay be captured, and a concentration series of mouse, cyno, or humanANGPTL3/8 complex may be injected over the chip surface at about 50uL/min for about 240 seconds with a dissociation time of about 800seconds. To determine kinetic parameters (e.g., k_(a), k_(d), K_(D))data is double-referenced and fit to a 1:1 binding model using BiacoreT200 Evaluation Software (GE Healthcare Bio-Sciences Corp.). Table 7,below, shows the binding of an anti-ANGPTL3/8 complex Ab having a LC ofSEQ ID NO:5 and a HC of SEQ ID NO:6 to various ANGPTL3/8 complexes fromdifferent species at pH 7.4 and temperature 25° C.

TABLE 7 Binding of Ab to Cross Species ANGPTL3/8 Complexes at pH 7.4 andTemperature 25° C. Species ANGPTL3/8 Complex Binding to Ab k_(a) (1/Ms)k_(d) (1/s) K_(D) (M) s.d. N Mouse 4.75 × 10⁵ 1.72 × 10⁻⁴ 3.66 × 10⁻¹⁰6.19 × 10⁻¹¹ 3 Cyno 4.91 × 10⁵ 9.91 × 10⁻⁵ 2.13 × 10⁻¹⁰ 6.30 × 10⁻¹¹ 3Human 4.77 × 10⁵ 6.31 × 10⁻⁵ 1.35 × 10⁻¹⁰ 2.69 × 10⁻¹¹ 2

In addition to the above anti-ANGPTL3/8 complex Ab having a LC of SEQ IDNO:5 and a HC of SEQ ID NO:6, Abs with LC variants having a D31Smutation (SEQ ID NO:21), a D33A mutation (SEQ ID NO:22) or a E99Qmutation (SEQ ID NO:25) and a HC of SEQ ID NO:6 are assayed for bindingto various ANGPTL3/8 complexes from different species at pH 7.4 andtemperature 25° C. (Tables 8-10).

TABLE 8 Binding of D31S Variant Ab to Cross Species ANGPTL3/8 Complexesat pH 7.4 and Temperature 25° C. Species ANGPTL3/8 Complex Binding toD31S Ab k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) s.d. N Mouse 4.96 × 10⁵ 1.08× 10⁻⁴ 2.18 × 10⁻¹⁰ 1.88 × 10⁻¹¹ 3 Cyno 4.62 × 10⁵ 1.68 × 10⁻⁴ 3.65 ×10⁻¹⁰ 1.50 × 10⁻¹¹ 3 Human 4.97 × 10⁵ 5.75 × 10⁻⁵ 1.16 × 10⁻¹⁰ 1.91 ×10⁻¹¹ 3

TABLE 9 Binding of D33A Variant Ab to Cross Species ANGPTL3/8 Complexesat pH 7.4 and Temperature 25° C. Species ANGPTL3/8 Complex Binding toD33A Ab k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) s.d. N Mouse 5.15 × 10⁵ 3.47× 10⁻⁴ 6.80 × 10⁻¹⁰ 7.07 × 10⁻¹¹ 3 Cyno 4.35 × 10⁵ 2.65 × 10⁻⁴ 6.09 ×10⁻¹⁰ 1.21 × 10⁻¹¹ 3 Human 4.57 × 10⁵ 9.19 × 10⁻⁵ 2.01 × 10⁻¹⁰ 1.75 ×10⁻¹¹ 3

TABLE 10 Binding of E99Q Variant Ab to Cross Species ANGPTL3/8 Complexesat pH 7.4 and Temperature 25° C. Species ANGPTL3/8 Complex Binding toE99Q Ab k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) s.d. N Mouse 5.96 × 10⁵ 1.51× 10⁻⁴ 2.54 × 10⁻¹⁰ 1.79 × 10⁻¹¹ 3 Cyno 6.20 × 10⁵ 7.23 × 10⁻⁵ 1.16 ×10⁻¹⁰ 8.58 × 10⁻¹² 3 Human 6.74 × 10⁵ 3.26 × 10⁻⁵ 4.84 × 10⁻¹¹ 6.50 ×10⁻¹² 3

Example 4 In Vitro Functional Activity LPL Assay

A cell based bioassay is used to assess the ability of theanti-ANGPTL3/8 complex Ab having a LC of SEQ ID NO:5 and a HC of SEQ IDNO:6 to de-repress ANGPTL3/8 purified protein inhibition of LPLactivity. The Ab's ANGPTL3/8 inhibitory activity is determined usingEnzChek™ Lipase Substrate (ThermoFisher). HEK293 cell lines expressinghuman, cynomolgus, mouse or rat LPL, and purified human, cynomolgus,mouse or rat ANGPTL3/8 protein. HEK293-LPL generation includes a humanembryonic cell line stably expressing human LPL, which is generated(HEK293-huLPL) utilizing standard methods. Briefly, human LPL is clonedinto a lentivirus plasmid with a CMV promoter and blasticidinresistance. The plasmid is used to generate a human LPL lentivirus usingViraPower Packaging Mix (Invitrogen). HEK293 cells are incubated withthe LPL lentivirus and clones resistant to blasticidin are selected. Aclone is chosen after confirming human LPL mRNA expression by qPCR andLPL activity utilizing the EnzChek™ substrate. This process is repeatedfor cynomolgus, mouse and rat LPL.

Methods are modified from those described in Basu et al. (Basu et al.(2011) J. Lipid Res. 52:826-832): (a) HEK293-LPL cells are added to ahalf area 96-well Poly-D-Lysine coated plate A (human), B (cynomolgus),C (mouse) or D (rat) at a density of 25000 cells/well and are incubatedovernight at 37° C., 5% CO₂. Ab is serially diluted nine times from astarting stock concentration to generate a ten-point CRC, and then addedto purified ANGPTL3/8 proteins (at the IC₈₀ concentration of 0.42 nM forhuman, 0.38 nM for cynomolgus, 0.13 nM for mouse, or 0.81 nM for rat) in96 well plates E (human), F (cynomolgus), G (mouse) or H (rat).

The media on the HEK293-LPL cells in plates A, B, C, and D are replacedwith the ANGPTL3/8 and Ab mixtures from plate E, F, G and H,respectively, and are incubated 1 hr at 37° C., 5% CO₂. 10 μl ofEnzChek™ Lipase Substrate (prepared at a concentration of 5μM in 0.05%Zwittergent (3-(N,N-Dimethyloctadecylammonio)propanesulfonate) (Sigma)is added to the cells, ANGPTL3/8 and Ab mixture in plates A, B, C and D.A plate reader is used to measure the fluorescence at 482 nm Excitationand 515 nm Emission with a 495 nm cutoff filter. The plates areincubated at 37° C., 5% CO2 in between time points. Relativefluorescence (directly proportional to LPL activity) is calculated bysubtracting the signal at 1 min from the signal at 31 min. The efficacyconcentration at which the Ab restored LPL activity 50% (EC₅₀) iscalculated utilizing Excel Fit. The EC₅₀ concentrations are shown inTable 11.

The percent derepression is calculated as follows:

=(RFU−RFU(MIN))/(RFU(MAX)−RFU(MIN))×100,

where MAX=cells alone (LPL) and MIN=cells (LPL)+ANGPTL3/8 CM(conditioned media).

The Ab generally has a low EC₅₀ and so is potent in de-repressing LPL.The Ab also has favorable % max de-repression of LPL.

TABLE 11 Ab EC₅₀ and % Max Derepression of LPL for Human, Cyno, Mouseand Rat LPL and Human, Cyno, Mouse and Rat ANGPTL3/8 Complex. LPL andANGPTL3/8 % Max Species Ab EC₅₀ (nM) Derepression of LPL human LPL andhuman 0.28 102% ANGPTL3/8 cyno LPL and cyno 1.31  96% ANGPTL3/8 mouseLPL and mouse 1.38 103% ANGPTL3/8 rat LPL and rat ANGPTL3/8 2.77 105%

In addition to the above anti-ANGPTL3/8 complex Ab having a LC of SEQ IDNO:5 and a HC of SEQ ID NO:6, Abs with LC variants having a D31Smutation (SEQ ID NO:21), a D33A mutation (SEQ ID NO:22), a D33T mutation(SEQ ID NO:23) or a E99Q mutation (SEQ ID NO:25) and a HC of SEQ ID NO:6are assayed to derepress ANGPTL3/8 purified protein inhibition of LPLactivity (Table 12).

TABLE 12 Variant Ab EC₅₀ and % Max Derepression of LPL for Human andMouse LPL and Human and Mouse ANGPTL3/8 Complex. D31S D33A D33T E99Q AbEC₅₀ Ab EC₅₀ Ab EC₅₀ Ab EC₅₀ (nM), (nM), (nM), (nM), LPL and % Max % Max% Max % Max ANGPTL3/8 Derepression Derepression DerepressionDerepression Species LPL LPL LPL LPL human LPL 0.86, 106% 1.14, 103%1.56, 105% 0.49, 106% and human ANGPTL3/8 mouse LPL and 1.79, 110% 4.07,106% 4.55, 114% 1.65, 110% mouse ANGPTL3/8

Example 5 In Vivo Triglyceride Response

The effect of an anti-ANGPTL3/8 complex Ab having a LC of SEQ ID NO:5and HC of SEQ ID NO:6 on serum TG is evaluated in mice transgenic forhuCETP and huApolipoprotein A1 (a). Blood is collected from the mice,and serum is isolated prior to the start of the experiment. TG ismeasured in serum samples using a Cobas® clinical chemistry analyzer(Roche). Animals are assigned into 5 groups of 20 to yield groups withsimilar serum TG averages. Each group of 20 is then further subdividedinto 4 groups of 5 with similar serum triglyceride averages. The Ab isadministered to mice by a single subcutaneous injection at 1 mg/kg(n=20), 3 mg/kg (n=20), 10 mg/kg (n=20), or 30 mg/kg (n=20) to 4separate groups of animals. A control antigen binding null isotypematched mAb is administered by a single subcutaneous injection at 30mg/kg(n=20) to a fifth group of animals.

Blood is collected from the animals 1 hour (n=5), 8 hours (n=5), 1 day(n=5), 2 days (n=5), 3 days (n=5), 7 days (n=5), 14 days (n=5) and 21days (n=5) after Ab administration. Blood is collected from subgroup Aanimals at times 1 hour and 3 days. Blood is collected from subgroup Banimals at times 8 hours and 7 days. Blood is collected from subgroup Canimals at times 1 day and 14 days. Blood is collected from subgroup Danimals at times 2 days and 21 days. Serum is prepared from the bloodand serum TG levels were measured using a Cobas® clinical chemistryanalyzer (Roche). Percent change of TG from the time matched isotypecontrol is calculated for each dose of Ab at each time point. Thecalculation for percent change is [(Rx Serum triglyceride−time matchedisotype control serum triglyceride)/(time matched isotype control serumtriglyceride)]×100. Data is shown in Table 13.

TABLE 13 Percent TG Lowering by Ab Compared to IgG Control of DifferentDoses of Ab at Different Time Points. Time Post 1 8 1 2 3 7 14 21 Dosehour hour day day day day day day  1 mg/kg −22 −33  −76* −66* −61*   41    6.4  −20   3 mg/kg −26 −71* −84* −77* −86* −25    19.7  −19  10 mg/kg−27 −62* −87* −89* −88* −88* −64.4* −28  30 mg/kg −23 −74* −92* −87*−93* −93* −89.1* −75* Dunnett's test is used for each data set tocompare each treatment group to a time matched control and a p-value of< 0.05 was considered statistically significant. Groups statisticallysignificant from their time matched controls are designated by a (*) inthe table.

The potency of the Ab having a LC of SEQ ID NO:5 and HC of SEQ ID NO:6at lowering TGs is favorable starting at Day 1 and the favorable effectis sustained until Day 21.

In addition to the above anti-ANGPTL3/8 complex Ab having a LC of SEQ IDNO:5 and a HC of SEQ ID NO:6, Abs with LC variants having a D31Smutation (SEQ ID NO:21), a D33A mutation (SEQ ID NO:22) or a E99Qmutation (SEQ ID NO:25) and a HC of SEQ ID NO:6 are assayed to evaluatechange in TGs relative to an IgG control in mice (Table 14).

TABLE 14 Percent TG Lowering Compared to IgG Control of Different Dosesof Variant Abs at Different Time Points. Time Post Dose 1 day 7 day 15day 21 day D31S −83* −38* −25  −12  3 mg/kg D31S −90* −91* −37* −21 10mg/kg D33A −74* −77* −34* −10  3 mg/kg D33A −78* −86* −76*  −60* 10mg/kg E99Q −82* −49* −5   15  3 mg/kg E99Q −86* −91* −61*    6 10 mg/kgDunnett's test is used for each data set to compare each treatment groupto a time matched control and a p-value of <0.05 was consideredstatistically significant. Groups statistically significant from theirtime matched controls are designated by a (*) in the table.

SEQUENCE LISTING

The following nucleic and amino acid sequences are referred to in thedisclosure above and are provided below for reference.

SEQ ID NO: 1atatatagagttaagaagtctaggtctgcttccagaagaaaacagttccacgttgcttgaaattgaaaatcaagataaaaatgttcacaattaagctccttctttttattgttcctctagttatttcctccagaattgatcaagacaattcatcatttgattctctatctccagagccaaaatcaagatttgctatgttagacgatgtaaaaattttagccaatggcctccttcagttgggacatggtcttaaagactttgtccataagacgaagggccaaattaatgacatatttcaaaaactcaacatatttgatcagtctttttatgatctatcgctgcaaaccagtgaaatcaaagaagaagaaaaggaactgagaagaactacatataaactacaagtcaaaaatgaagaggtaaagaatatgtcacttgaactcaactcaaaacttgaaagcctcctagaagaaaaaattctacttcaacaaaaagtgaaatatttagaagagcaactaactaacttaattcaaaatcaacctgaaactccagaacacccagaagtaacttcacttaaaacttttgtagaaaaacaagataatagcatcaaagaccttctccagaccgtggaagaccaatataaacaattaaaccaacagcatagtcaaataaaagaaatagaaaatcagctcagaaggactagtattcaagaacccacagaaatttctctatcttccaagccaagagcaccaagaactactccctttcttcagttgaatgaaataagaaatgtaaaacatgatggcattcctgctgaatgtaccaccatttataacagaggtgaacatacaagtggcatgtatgccatcagacccagcaactctcaagtttttcatgtctactgtgatgttatatcaggtagtccatggacattaattcaacatcgaatagatggatcacaaaacttcaatgaaacgtgggagaactacaaatatggttttgggaggcttgatggagaattttggttgggcctagagaagatatactccatagtgaagcaatctaattatgttttacgaattgagttggaagactggaaagacaacaaacattatattgaatattctttttacttgggaaatcacgaaaccaactatacgctacatctagttgcgattactggcaatgtccccaatgcaatcccggaaaacaaagatttggtgttttctacttgggatcacaaagcaaaaggacacttcaactgtccagagggttattcaggaggctggtggtggcatgatgagtgtggagaaaacaacctaaatggtaaatataacaaaccaagagcaaaatctaagccagagaggagaagaggattatcttggaagtctcaaaatggaaggttatactctataaaatcaaccaaaatgttgatccatccaacagattcagaaagctttgaatgaactgaggcaaatttaaaaggcaataatttaaacattaacctcattccaagttaatgtggtctaataatctggtattaaatccttaagagaaagcttgagaaatagattttttttatcttaaagtcactgtctatttaagattaaacatacaatcacataaccttaaagaataccgtttacatttctcaatcaaaattcttataatactatttgttttaaattttgtgatgtgggaatcaattttagatggtcacaatctagattataatcaataggtgaacttattaaataacttttctaaataaaaaatttagagacttttattttaaaaggcatcatatgagctaatatcacaactttcccagtttaaaaaactagtactcttgttaaaactctaaacttgactaaatacagaggactggtaattgtacagttcttaaatgttgtagtattaatttcaaaactaaaaatcgtcagcacagagtatgtgtaaaaatctgtaatacaaatttttaaactgatgcttcattttgctacaaaataatttggagtaaatgtttgatatgatttatttatgaaacctaatgaagcagaattaaatactgtattaaaataagttcgctgtctttaaacaaatggagatgactactaagtcacattgactttaacatgaggtatcactataccttatttgttaaaatatatactgtatacattttatatattttaacacttaatactatgaaaacaaataattgtaaaggaatcttgtcagattacagtaagaatgaacatatttgtggcatcgagttaaagtttatatttcccctaaatatgctgtgattctaatacattcgtgtaggttttcaagtagaaataaacctcgtaacaagttactgaacgtttaaacagcctgacaagcatgtatatatgtttaaaattcaataaacaaagacccagtccctaaattatagaaatttaaattattcttgcatgtttatcgacatcacaacagatccctaaatccctaaatccctaaagattagatacaaattttttaccacagtatcacttgtcagaatttatttttaaatatgattttttaaaactgccagtaagaaattttaaattaaacccatttgttaaaggatatagtgcccaagttatatggtgacctacctttgtcaatacttagcattatgtatttcaaattatccaatatacatgtcatatatatttttatatgtcacatatataaaagatatgtatgatctatgtgaatcctaagtaaatattttgttccagaaaagtacaaaataataaaggtaaaaataatctataattttcaggaccacagactaagctgtcgaaattaacgctgatttttttagggccagaataccaaaatggctcctctcttcccccaaaattggacaatttcaaatgcaaaataattcattatttaatatatgagttg cttcctctattSEQ ID NO: 2 MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSI KSTKMLIHPTDSESFESEQ ID NO: 3ataccttagaccctcagtcatgccagtgcctgctctgtgcctgctctgggccctggcaatggtgacccggcctgcctcagcggcccccatgggcggcccagaactggcacagcatgaggagctgaccctgctcttccatgggaccctgcagctgggccaggccctcaacggtgtgtacaggaccacggagggacggctgacaaaggccaggaacagcctgggtctctatggccgcacaatagaactcctggggcaggaggtcagccggggccgggatgcagcccaggaacttcgggcaagcctgttggagactcagatggaggaggatattctgcagctgcaggcagaggccacagctgaggtgctgggggaggtggcccaggcacagaaggtgctacgggacagcgtgcagcggctagaagtccagctgaggagcgcctggctgggccctgcctaccgagaatttgaggtcttaaaggctcacgctgacaagcagagccacatcctatgggccctcacaggccacgtgcagcggcagaggcgggagatggtggcacagcagcatcggctgcgacagatccaggagagactccacacagcggcgctcccagcctgaatctgcctggatggaactgaggaccaatcatgctgcaaggaacacttccacgccccgtgaggcccctgtgcagggaggagctgcctgttcactgggatcagccagggcgccgggccccacttctgagcacagagcagagacagacgcaggcggggacaaaggcagaggatgtagccccattggggaggggtggaggaaggacatgtaccctttcatgcctacacacccctcattaaagcagagtcgtggcatctcaaaaaaaaaaaaaaaaa SEQ ID NO: 4MPVPALCLLWALAMVTRPASAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQIQERLHTAALPA SEQ ID NO: 5DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSENRGEC SEQ ID NO: 6QVTLKESGPTLVKPTQTLTLTCTFSGFSLSISGVGVGWIRQPPGKALEWLALIYRNDDKRYSPSLKSRLTITKDTSKNQVVLTLTNMDPVDTATYYCARTYSSGWYGNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGSEQ ID NO: 7gatattgtgatgacccagacccccctgtctctgcctgtcactccgggggaaccggcctcgatctcatgccggtcgagccagtccctgctggactccgatgacgggaacacttatttggattggtacctccaaaagcctggacagagcccgcagctcctgatctacatgctgtcctaccgggcctccggagtgccagaccgcttctcgggaagcggctccggtaccgacttcacactgaagatctcccgcgtggaagctgaggacgtgggcatctactactgtatgcaaagaatcgagttccccctcaccttcggcggcgggactaaggtcgagattaagagaaccgtggccgcaccatccgtgttcatttttcccccgtccgatgaacagctgaagtccggaaccgcctccgtcgtgtgcctgctcaacaacttctacccgagggaagcgaaagtgcagtggaaagtggacaatgcgctgcagtccggaaactcccaagagtccgtgaccgaacaggactccaaggactcaacctactcgctgagctcaacgctgaccctgagcaaggccgactacgagaagcacaaggtctacgcctgcgaagtgacccatcagggtttgagctcgcccgtgaccaagtccttcaaccggggagagtgcSEQ ID NO: 8caagtcacattgaaggagagcggtccgaccctggtcaagccgactcagaccctgaccctgacgtgcactttctcgggcttctcattgtccatttctggagtgggcgtgggatggatcagacagcccccggggaaggccctcgagtggctcgcgctgatctaccgcaacgacgacaaacgctactccccctcactgaaatcccggctgaccatcactaaggatacgtccaagaaccaggtcgtgttgaccctcaccaacatggatcccgtggatactgccacctactattgtgcacggacctatagcagcggttggtacggaaactggttcgacccgtggggccagggaactcttgtgacggtgtcctccgcaagcaccaagggtccttctgtgttccccctggcgccgtgctcgcggagcacacagagtccaccgccgccctcggctgccttgtgaaggactacttcccggagccagtcaccgtgtcctggaacagcggggccctgacttccggcgtgcacaccttccctgcggtgctgcagagctcaggcctctattcgctgtcatccgtcgtgaccgtgccttcctcgtccctgggcactaagacctacacttgcaacgtggaccataagcccagcaacaccaaagtggacaagagagtggaatccaaatacggaccgccatgtccgccctgccccgccccggaagctgccgggggacccagcgtgttcctgttcccacctaagccgaaggacactctgatgatctcaaggactcccgaagtcacttgcgtggtcgtggacgtgtcccaggaggaccccgaagtccagtttaattggtacgtggatggtgtcgaggtccacaacgccaagaccaagcctcgcgaggaacagttcaattccacctaccgggtcgtgtccgtcctgaccgtgctgcatcaggactggctgaacggaaaggagtacaagtgcaaagtgtccaacaagggactcccttcctccatcgaaaagaccatcagcaaggccaagggccagcctcgcgaaccacaagtctacaccagcccccatcgcaagaggaaatgaccaagaaccaagtgtcgctgacatgcctcgtcaagggattctacccgtcggatattgcggtggaatgggagtccaacggacagcccgagaacaactacaagaccaccccgccggtgttggactccgacggctcctttttcctgtactcccggctcactgtggacaagtcgcggtggcaggaggggaacgtgttctcctgttccgtgatgcacgaagctctgcacaaccactacacccagaagtcgctgagcctctcactggga SEQ ID NO: 9DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEI K SEQ ID NO: 10QVTLKESGPTLVKPTQTLTLTCTFSGFSLSISGVGVGWIRQPPGKALEWLALIYRNDDKRYSPSLKSRLTITKDTSKNQVVLTLTNMDPVDTATYYCARTYSSGWYGNW FDPWGQGTLVTVSSSEQ ID NO: 11 RSSQSLLDSDDGNTYLD SEQ ID NO: 12 YMLSYRAS SEQ ID NO: 13MQRIEFPLT SEQ ID NO: 14 TFSGFSLSISGVGVG SEQ ID NO: 15 LIYRNDDKRYSPSLKSSEQ ID NO: 16 ARTYSSGWYGNWFDP SEQ ID NO: 17MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFEAAADYKDDDDK SEQ ID NO: 18METDTLLLWVLLLWVPGSTGDHHHHHHDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLAPAPAPAPAPAPAPAPAPAPLEVLFQGPGRAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQI QERLHTAALPASEQ ID NO: 19 SRIDQDNSSFDSLSPEPKSRFAMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE AAADYKDDDDKSEQ ID NO: 20 GPGRAAPMGGPELAQHEELTLLFHGTLQLGQALNGVYRTTEGRLTKARNSLGLYGRTIELLGQEVSRGRDAAQELRASLLETQMEEDILQLQAEATAEVLGEVAQAQKVLRDSVQRLEVQLRSAWLGPAYREFEVLKAHADKQSHILWALTGHVQRQRREMVAQQHRLRQIQERLHTAALPA SEQ ID NO: 21DIVMTQTPLSLPVTPGEPASISCRSSQSLLSSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 22DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSADGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 23DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSTDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 24DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIEFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 25DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSPQLLIYMLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQRIQFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

The invention claimed is:
 1. A polynucleotide comprising a nucleic acidsequence encoding a polypeptide of SEQ ID NO:17 or a polypeptide of SEQID NO:18.
 2. A polynucleotide comprising a nucleic acid sequenceencoding a polypeptide of SEQ ID NO:19 and a polypeptide of SEQ IDNO:20.
 3. An expression cassette comprising the polynucleotide ofclaim
 1. 4. A host cell comprising the expression cassette of claim 3.5. The host cell of claim 4, wherein the host cell is a mammalian cell.6. A polypeptide comprising an amino acid sequence of SEQ ID NO:17 or19.
 7. A polypeptide comprising an amino acid sequence of SEQ ID NO:18or
 20. 8. An angiopoietin-like peptide (ANGPTL)3/8 complex comprising apolypeptide having an amino acid sequence of SEQ ID NO:19 and apolypeptide having an amino acid sequence of SEQ ID NO:20.